Chromosome replication and transmission are essential for the inheritance of genetic traits, but the mechanisms responsible for these processes remain poorly understood in multicellular eukaryotes. The centromere, which appears as a constriction in metaphase chromosomes, is required for kinetochore formation, which serves as the key attachment site to the spindle during mitosis and meiosis. Defects in centromere or kinetochore function result in aneuploidy, which is a hallmark of human cancers and is responsible for many birth defects. A pressing question in the centromere field today is how centromere identity is propagated from one generation to the next in multicellular eukaryotes. Elucidating the determinants of centromere identity, propagation and function requires identification of the gene products that promote centromere formation and function, and determining the mechanisms responsible for assembly of centromeric chromatin. The multifaceted approaches required to address these complex questions in higher eukaryotes are likely to succeed in Drosophila. There is over a century of experimental analyses and biological information that facilitates sophisticated in vivo analyses. Here, we propose genetic, molecular, cell biological and biochemical experiments designed to identify and characterize gene products that promote the assembly and propagation of centromeric chromatin, and to determine their properties and functions. Our entry point into centromeric chromatin is a conserved histone H3-like protein (CID, for Centromere IDentifier) that localizes exclusively to functional centromeres. We will capitalize on results obtained in the previous funding period, which identified key regulators of CID localization and assembly, as well as a link between cell cycle regulation and centromere formation. We propose to investigate the interactions and functions of centromere regulators to elucidate molecular mechanisms of centromere assembly and its regulation through the cell cycle. These studies will address specific hypotheses and provide the groundwork for future analysis of inheritance and centromere function in Drosophila and other higher eukaryotes, such as humans. PUBLIC HEALTH RELEVANCE: Human cancers uniformly contain massive numbers of chromosome rearrangements and other consequences of genome instability. Centromere dysfunction is one cause of genome instability, and misexpression of the centromeric histone we study has been observed in human colon and breast tumors. The results of this project will provide key information about the normal regulation of centromere assembly, which will be important to the development of cancer diagnostic and treatment tools.